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UNIVERSITI PUTRA MALAYSIA
CHARACTERISATION OF SALMONELLA ENTERITIDIS ISOLATES FROM HUMANS, ANIMALS AND ENVIRONMENTAL SOURCES.
MAIMUNAH BT. MUSTAKIM
FSMB 1998 2
CHARACTERISATION OF SALMONELLA ENTERITIDIS ISOLATES FROM HUMANS, ANIMALS AND ENVIRONMENTAL SOURCES.
By
MAIMUNAH BT. MUSTAKIM
Thesis submitted in Fulfilment of the Requirements for the Degree of Master of Science in the Faculty of
Food Science and Biotechnology, Universiti Putra Malaysia.
August 1 998
ACKNOWLEDGEMENTS
The magnitude and scope of work involved in this research
required the assistance of numerous people, to whom the author
indebted, however great or small their respective contribution.
F irst, the author expresses her great appreciation to Dr. Son
Radu, the Committee Chairman, for his kindness, wi l l ingness to help,
and encouraging advise throughout the research work.
Sincere appreciation was extended to Professor Dr. Gulam Rusul
Rahmat Ali and Dr. Zaiton Hassan, the Committee Members,
especially for their suggestions and critical reviews of the thesis.
Very special thanks dedicated to al l her friends in Microbiology
Laboratory : Sahi lah, Kak Endang, Zainuri , Zunita, Dayang Fredal ina,
Loke Chui Fung, Rozila, Wong Wooi Koon, Yuherman, Samuel, Kami l
and Razal i .
The family : beloved mother, husband and chi ldren, their never
ending devotions and moral support throughout her education were
gratefully acknowledged.
i i i
TABLE OF CONTENTS
Page ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . i i i L IST OF TABLES . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . v i i L IST OF F IGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . ix L IST OF PLATES . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . x ABSTRACT . . . . . . . . , . . . . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i i i
CHAPTER
I I
I I I
GENERAL INTRODUCTION . . . . . . . . . . . . . . , . . . . . . ... . . . . 1
LITERATURE REViEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Genus Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Salmonella in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Salmonella in Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella i n Natural Environment . . . . . . . . . . .
Worldwide Problem of Salmonel losis . . . . . . . . .
The Systematics of Salmonella . . . . . . . . . . . . . . .. . . . . . . . . . . . . . D ivisions of Salmonella into Serotype . . .
Subdivisions of Salmonella Serovars . . . . . .
Appl ication of Molecular Techniques in Subdivision of Salmonella Serovars . . . . . , . . . . . . . . . . . . . .
Plasmid Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
R Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conjugal Transfer of R Plasmids . . . . . . . . .
Chromosomal DNA Analysis by Pulse-field Gel Electrophoresis . . . . . . . . . . . . . . . . , . . . . . . . . . . .
Pulsed-field Gel Electrophoresis . . . . . . . . . . . .
Restriction Endonuclease for Chromosomal DNA Analysis . . . . . . . . . . . . . . . . . . . .
BIOTYPING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . .
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . .
Collection and Maintenance . . . . . . . . . . . . . . . . . .
Biotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .
Results . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . .
Biotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iv
7 7 8
1 0 1 4 1 6 1 7 1 8 24 27
33 34 38 39
42 43
45
47 47 50 50 51 55 55
Biotyping for S. enteritidis Phage-typed Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
IV PLASMID PROFILING AND ANTIMICROBIAL RESISTANCE OF S. ENTERITIDIS ISOLATES . . . 58 Introduction . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Bacterial Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Isolation of Plasmid DNA . . . . . . . . , . . . . . . . . . . . . 60 Detecting Plasmid DNA by Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Gel Visual isation and Photography . . . . . . . . . . . . 62 Plasmid DNA Size Reference . . . . . . . ; . . . . . . . 63
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Plasmid Profiles Analysis of Salmonella enteritidis Isolates . . . . . . . . . . . . . . . . . . . 65 Antimicrobial Resistance in S. enteritidis. . . . . 73 Relationships between Antibiotic Resistance and Possession of P lasmid . . . . . 75 Subdivision of S. enteritidis Phage-typed Strains by Plasmid Profi l ing and Antibiotic Resistance Patterns . . . . . . . . . . . . . . . . . . . 75
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
V CONJUGATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . 82 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Bacterial Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Conjugation of Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Preparation of Selective Media . . . . . . . . . . . . . . . 85 Determination of Plasmid(s) in Transconjugant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Discussion ... . . .. " ... ... ... ... ... ... ... ... ... ................ 90
VI RESTRICTION ENDONUCLEASE DIGESTION POLYMORPHISM OF S. ENTERITIDIS BY PULSED-FIELD GEL ELECTROPHORESIS . . . . . . 93 Introduction . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" . . . . . . 93 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Preparation of H igh Molecular Weight DNA by Agarose Embedding Bacterial Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Restriction Enzymes Digestion of DNA Plugs . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
v
Detecting Restriction Endonuclease Digestion by Pulsed-field Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Restriction Endonucleases . . . ... . .. . .. ... ... .... 98 Genome S ize Measurement . . . ... ... ... ......... 99 Data Analysis . . . ... ... ... ... .. . ... ... ... ... . .. . .. . . . . 99
Result . . . . . . . . . . . . . . , ... ...... ......... ... '" ... ... ... ... ... 1 02 Restriction Polymorphism of Chromosomal DNA of the S. enteritidis Isolates . . . . . . . . . . . . . . . . . 1 02 Distribution of Restriction Polymorph isms in S. enteritidis Isolates. . . . . . ... ... ... ... .......... 1 1 0 Variations of PFGE Patterns in Humans, Animals and Environmental Isolates . . . . . . . . . 1 1 2 Subdivision of S . enteritidis Phage-typed Strains by REDP-PFGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1"1'9 Characterisation of S . enteritidis Isolates by Restriction Polymorphism of the Chromosomal DNA. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . 1 1 9 Factors Influencing the Separation of DNA Fragments by Pulsed-field Gel Electrophoresis . . . . . . . . . . . . . . . .................... ... ... 1 20 Comparison of the Restriction Endonuclease Patterns in Humans, Animals and Environmental Sources . . . ... ... 1 2 1 Genomic Size of S . enteritidis. . . . . . . . . . . . . . . . . . . . . 1 23 Characterisation of S . enteritidis Phage-typed Strains by REDP-PFGE . . . . . . . . . . . . . . . . . . . 1 23
VI I GENERAL DISCUSS ION AND CONCLUSION . . . . . 1 26
REFERENCES . . . ... ... . .. .. . . . . . .. .. . ... ... ... ... . . . ... ... ... ... ... .... 1 31
APPEND IX . . . .... . . . . . . . . . . . ....... ....... . . ... . . . . . ....... ......... . . ... ............. 1 39
VITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" ... ... . . . ... ... . . . ... ... ... ... ... . . . . . 1 49
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LIST OF TABLES
Table Page
1 Source of Food Contamination . . . . . . . . . . . . . . . . . . . . . . . . 1 2
2 Differentiation of Salmonella with other genus of the Enterobactericeae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3 Abbreviated Kaufmann-White Scheme of Salmonella Serovar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4 Determination of S. typhimurium Phage Type . . . . . . 30
5 Biotyping Scheme for Salmonella enteritidis . . . . . . . . 49
6 The 1 05 S. enteritidis Isolates . . . . . . . . . . . . . . . . . . . . . . . . 50
7 Biotyping of S. enteritidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
8 Biotyping of S. enteritidis Phage-typed Strains . . . . . 56
9 Plasmid Profi les in Salmonella enteritidis . . . . . . . . . . . 66
1 0 Plasmid Classes of S. enteritidis Isolates . . . . , . . . . . . . 66
1 1 Antimicrobial Resistance Patterns of S . enteritidis from Various Sources . . , . . . . . . . . , . . . . . . . . , . . . . . . . . . . . . . . . . 74
1 2 Relationships between Antibiotic Resistance Patterns and Plasmid Profi les . . . . . . . . . . . . . . . . . . . . . . . . 76
1 3 Relationships Among Phage-type, P lasmid Profi les and Antibiotic Resistance Patterns . . . . . . . . . 77
1 4 The Abi l ity of Gene Transfer Among S . enteritidis Isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
1 5 Characterization of Transconjugants . . . . . . . . . . . . . . . 89
1 6 REDP-PFGE of S . enteritidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1
1 7 Interpretation of Pulse-field Gel Electrophoresis Patterns . . , ' " . . . . . , . . . ' " . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 3
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1 8 Simi larity Index ( F Values) for S . enteritidis Strains from Xbal Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 1 1 5
1 9 Simi larity Index (F Values) for S . enteritidis Strains from Spel Digestion . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . , . . 1 1 6
20 Subdivision of S. enteritidis Phage-typed Strains by REDP-PFGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8
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LIST OF FIGURES
Figure Page
1 Comparison of Reported Cases of Salmonellosis Excluding Typhoid Fever between the Year 1 941 to 1 985 and 1 988 to 1 995 . . . . . . . . . . . . ... . . . . . . . . . . . . . . . 3
2 Polysaccharide Structure of the B Antigenic Group of Salmonella . . . . . . . . . . . . . . . . . . . . , . . . . . . . . , . . . . . . . 25
3 Physical Forms of Plasm ids . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4 Graph of E lectrophoretic Mobi l ity for DNA S ize Standard of V51 7 ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
5 Graph of Electrophoretic Mobi l ity for DNA S ize Standard of A, Concatamer . . . . . . . . . . . . . . . . . . . . . ' " . . . . . . 1 00
6 A Schematic Representative of Xbal-PFGE F ingerprints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 03
7 A Schematic Representative of Spel-PFGE Fingerprints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . 1 07
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LIST OF PLATES
Plate Page
1 E lectron micrograph of P lasmid DNA . . . . . . . . . . . . . . . 36
2 Electron micrograph of Bacterial Conjugation . . . . 40
3 Plasmid Patterns of S. enteritidis Isolates from Several Animal Sources . . . . . . . , . . . . . . . . . . . , . . . . . . . . . . . , . 69
4 Plasmid Patterns of S. enteritidis Isolates from Several Animal Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 70
5 Plasmid Patterns of S. enteritidis Isolates from Human Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1
6 Plasmid Patterns of S. enteritidis Isolates from Human Sources . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . 72
7 Plasmids in Transconjugant of S . enteritidis Isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
8 REDP-Xbal of S . enteritidis Isolates . . . . . . . . . . . . . . . . . . 1 05
9 REDP-Xbal of S . enteritidis Isolates . . . . . . . . . . . . ' " . . . 1 06
1 0 REDP-Spel of S. enteritidis Isolates . . . . . , . . . . . . . . . . . , 108
1 1 RED P-Spel of S. enteritidis Isolates . . . . . , . . . . . . . . . . . , 1 09
x
Abstract of thesis presented to the Senate of Universit i Putra Malaysia in fulfi lment of the requirements for the degree of Master of Science.
CHARACTERISATION OF SALMONELLA ENTERITIDIS ISOLATES FROM H UMANS, ANIMALS AND ENVIRONMENTAL SOU RCES
By
MAIMUNAH MUSTAKIM
August 1 998
Chairman Dr. Son Radu
Faculty : Food Science and Biotechnology
One hundred and five strains of Salmonella enteritidis isolated
from five different sources including humans, food animals, avian, wi ld
animals and envifOnments were studied. All isolates were
characterised for their biotype, plasmid profi les and resistance to
antibiotics. Biotyping of the isolates subdivided them into two biotypes
i .e., A and E . Subdivision of the isolates by plasmid profi l ing identified
fifteen plasmid patterns i . e. , PPO to PP1 4. Plasmid pattern P P 1 4 was
the most common pattern encountered in 47 (44%) of the total isolates
from various sources. Resistance to antibiotics was not uncommon
with 1 04 (99%) of the isolates were resistant to more than one
antibiotics. Subdivision by antibiotic resistance pattern exhibited ten
antibiograms designated as R1 to R1 0. A total of 87 (82%) of
xi
the isolates fel l into resistant pattern R 1 which showed resistant
towards erythromycin, penici l l i n G and triple sulpha. In conjug�tions
studies, the abi l ity of transferring antibiotic resistance was
demonstrated in the transconjugants with the concomitant transfer of
the respective p lasmid and resistant to tetracycl ine. S ixty seven
representative strains of S. enteritidis studied for restriction
endonuclease digestion polymorphism by - pulsed-field gel
electrophoresis (REDP-PFGE) exhibited different PFGE fragment
patterns. Digestion with low-frequency-cleavage restriction
endonucleases, i . e . , Xbal ( 5'- TCTAGA-3' )and Spel (5'-ACTAGT-3')
produced five and three restriction patterns respectively. Inter
relationships of the PFGE patterns among the isolates could be
determined by the calculation of simi larity index, F. The F values
grouped the strains as closely related, unrelated or ind istinguishable.
The phage-typed strains of S. enteritidis were subdivided into one
biotypes, eleven p lasmid profi les, six resistance patterns and seven
restriction polymorph isms. In general , characterisation by DNA-based
method of plasmid profil ing gave more discrimination compared to
other methods. These methods also provide effective adjunct to
antibiotic resistance pattern and phage typing for subdivision of S.
enteritidis.
xii
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Master Sains.
PENCIRIAN PENCILAN SALMONELLA ENTERITIDIS DARI SUMBERSUMBER MANUSIA, HAIWAN DAN PERSEKITARAN
Oleh
MAIMUNAH MUSTAKIM
Ogos 1 998
Pengerusi : Dr. Son Radu
Fakulti : Sains Makanan dan Bioteknologi
Seratus l ima penci lan Salmonella enteritidis diperolehi dari
pelbagai sumber termasuk manusia, haiwan ternakan, beburung,
haiwan l iar dan persekitaran telah dikaji . Kesemua penci lan itu
dicirikan secara biotip , pemprofi lan plasmid dan sifat kerintangan
kepada antibiotik. Secara b iotip , pencilan itu telah dibahagikan kepada
dua iaitu biotip A dan E. Pembahagian secara plasmid pemprofi lan
menunjukkan penci lan itu boleh dibezakan dengan l ima belas corak
plasmid iaitu PPO - PP1 4. Corak PP1 4 tersebar secara meluas di
kalangan sumber-sumber yang dikaj i . Kerintangan kepada antibiotik
adalah perkara biasa bagi penci lan dengan 1 04 (99%) penci lan
adalah rintang kepada sekurang-kurangnya satu jenis antibiotik.
Pembahagian secara corak kerintangan menghasi lkan sepuluh corak
iaitu R1 h ingga R1 0. Sebanyak 87 (82.9%) penci lan itu termasuk di
xiii
dalam corak R 1 yang menunjukkan kerintangan kepada eritromicin,
penici l l in G dan 'triple-sulpha' .Di dalam kajian konjugasi, kebo.lehan
memindahkan s ifat-sifat kerintangan telah ditunjukkan di dalam
transkonjugan di mana sifat kerintangan telah dipindahkan bersama
sama dengan plasmid. Kajian ke atas enampuluh tujuh waki l isolat
isolat dari pelbagai sumber secara pol imorfisma penghadaman enzim
restriksi yang dihasi lkan melalui e lectrophoresis gel "pulse-field"
menunjukkan corak-corak P FGE yang berbeza. Penghadaman dengan
enzim restriksi pemotongan-frekuensi-rendah iaitu Xbal ( 5'-TCTAGA-
3') dan Spe l (5'-ACTAGT-3') masing-masing menghasi lkan l ima dan
tiga corak restriksi. Hubung-kait strain-strain di dalam sumber masing
masing boleh dikira dari indeks persamaan, F. Dari ni lai F itu, strain
strain dikumpulkan kepada berhubungrapat, tiada perhubungan atau
sama di antara satu sama lain. Pembahagian strain-strain fajtip
menghasi lkan satu corak biotip, sebelas corak plasmid dan tujuh
polimorfisma penghadaman enzim restriksi . Pada amnya, pencirian
dengan kaedah-kaedah berasaskan DNA menunjukkan lebih banyak
perbezaan di kalangan strain-strain. Kaedah tersebut juga sangat
berfaedah untuk digunakan bersama-sama kaedah antibiogram dan
fajtip di dalam pembahagian strain-strain S. enteritidis.
xiv
CHAPTER I
GENERAL INTRODUCTION
Typhoid, paratyphoid and salmonellosis are col lective terms
associated to infection and fevers caused by Salmonella species.
Salmonella typhi, the etiologic agent of typhoid fever has long been
threatened and led serious i l lness to human population in both
developed and third-world countries. The World Health Organisation
(WHO) has estimated that, annually, there are 1 6.6 mi l l ion cases of
typhoid fever with nearly 600,000 deaths, and 1 .3 bi l l ion cases of
acute gastroenteritis due to non-typhoidal salmonellosis with 3 mi l l ion
deaths. In developing countries, contaminated water supply due to
improper disposal of human and animal wastes is the usual mode for
transmission of the disease.
Typhoid fever was recognised by cl inical signs and symptoms
before the advent of bacteriological era. Beginning with the isolation
of Bacillus cholerae-suis from pigs with hog cholera in 1 885 by Salmon
and Smith, fol lowed by isolation of simi lar bacteria from foodborne
intoxication by Gaffky and Paak in 1 885 and Gartner in1 888 (Le Minor,
1
2
1 992) or animal disease by Loeffler in 1 892, those baci l lus were then
created a genus and named Salmonella by Lignieres in 1 900.
Salmonel la infection has been notified as a g lobal problem
ranking first or second as a cause of food and waterborne diseases
(Gangarosa, 1 980). In the United States for example, incidence of
sal monel losis increased gradually from below 1 000 cases in 1 941 to
70,000 cases in 1 985. The number has remained between 40,000 to
50,000 cases from 1 988 to 1 995 [Centre for Disease Contro l , 1 996;
Figure 1 (8)]. The epidemiological pattern of salmonel la infections has
changed with time, place and environmental conditions. These
changes have resulted due to the changes in food habits in the
community as wel l as changes in food production technology. Also, the
emergence of drug resistant salmonel la made the situation to be more
compl icated.
As Salmonel la problems have caused unnecessary suffering,
death and great economic losses which have led the public health
authorities in several countries to institute survei l lance programmes in
an attempt to control the rapidly increasing incidence of the disease. In
the United States, Salmonella survei l lance was instituted by Centre for
Disease Control (CDC) Atlanta, Georgia in 1 966 as a co-operative
undertaking by 50 states. In England and Wales, a systematic
col lection of information on salmonella infections was started in 1 949
70000
60000
50000
40000
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20000
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(A)
O+-���--�-r--��--'-��� 1941 1945 1951 1955 1961 1965 1971 1975 1981 1985
50,000 45,000 40,000 35,000 30,000 25,000 20,000 15,000 10,000
(8)
5,000 O+-�---r--���-r--'-� 1988 1989 1990 1991 1992 1993 1994 1995
Figure 1 : Comparison of Reported Cases of Salmonellosis excluding Typhoid Fever between the Year 1 941 to 1 985 (A) and 1 988 to 1 995 (8).
3
( Source : A - Salmonella, 1 992. 8 - Centre for Disease Control and Prevention, 1 996)
4
by Salmonella Subcommittee of the Publ ic Health Laboratory
Services. Survei l lance program for typhoid in Malaysia is managed by
the Epidemiology Division, Ministry of Health. General ly,
salmonel la survei l lance programme implemented in many countries
over the world are important in identifying outbreaks, high risk
foods and the mechanism by which transmission occurs.
The genus Salmonella belongs to the fam i ly Enterobactericeae
and comprises of more than 2,200 serotypes. Besides its medical
importance, this genus has attracted the interest of a great number of
scientists (bacteriologists, immunologists, etc. ) for its wide range of
variations in the classification systems. These organisms have been
subdivided in many ways especial ly for epidemiological purposes on
the basis of biochemical reactions, serological analysis or further
ampl ified by phage typing. Based on biochemical characteristics,
Kauffmann subdivided the genera into four major groups: I , I I , I I I and IV
(Le Minor, 1 992) . Then they were further differentiated into seven
subspecies i .e . , I , I I , ilia, I I Ib , IV, V and VI by studies of the thermal
stabi l ity of hybrids (Le Minor, 1 992) . Description of serological
identification based on the 0 (somatic), VI (capsular) and H (flagel lar)
antigens distinguished the strains into more than 2 ,200 serotypes
(Kauffmann and White scheme). In 1 938, Craigie and Yen introduced
phage typing for S. typhi which proved to be a valuable and sensitive
tool in the control of typhoid fever. The abil ity of phage to distinguished
5
varieties among identical serotypes led to the development and
acceptance of phage typing as a significant epidemiolog ical
procedure. With the advent of DNA technology era, several molecular
approaches for bacterial identification have been employed
in Salmonella. The DNA-related techniques such as plasmid analysis ,
restriction endonuclease digestion polymorphism (REDP) , ribotyping
and polymerase chain reaction (PCR) were able to identify individual
strains with remarkable precision.
Objectives
Nontyphoidal salmonellosis has been recognised as the most
frequent foodborne infection world-wide. Incidence of foodborne
outbreaks in recent years observed the emergence of Salmonella
enteritidis as the most commonly isolated serotype in many parts of the
world. For instance , the isolation frequency of S. enteritidis from
humans in Britain, Greece, Spain and the United States showed a
significant increase of more than 50% between 1 980 and 1 994 (Ridley
et a/. , 1 996 ; Vatopoulos et a/. , 1 994; Anon, 1 991 and Centre for
Disease Contro l , 1 993).
Due to the recent prominence of S. enteritidis, identification of the
strain is essential for an effective investigation of the outbreak cases.
Combination of conventional as wel l as molecular typing methods
6
have been employed for a precise differentiation of the individual
strain. In the present work, S. enteritidis strains which are multiply
resistant to antimicrobial agents isolated from animals, humans and
environmental sources were characterised by using conventional and
molecular typing methods.
The specific objectives of the present investigation were as
fol lows:
1 . To carry out biochemical tests for biotyping analysis.
2. To characterised the strains by plasmid content, antimicrobial
resistant patterns and their correlation.
3. To study the abi l ity to transfer resistance by conjugation studies.
4. To characterise the representative strains of S. enteritidis by
restriction endonuclease digestion polymorphism generated by pulsed
field gel electrophoresis (REDP-PFGE).
5. To apply the REDP-PFGE technique in determining the genome
size.
6. To subdivide the S. enteritidis phage typed strains by biotyping,
plasmid profi l ing, antibiotic resistance patterns and REDP-PFGE
methods.
CHAPTER II
LITERATURE REVIEW
Introduction
The Salmonella was first coined by Lignieres in 1 900 as an
honour to D .E . Salmon, an American bacteriologist who reported on
"the hog cholera group of bacteria" in swine. These gram-negative rod
shaped bacteria are confirmed to be the member of the fami ly
Enterobacteriaceae and were assigned to genus I I I in the fami ly (Le
Minor, 1 984). They were characterised as moti le with petrichous
flagel la, facultatively anaerobic and usual ly produced gas from g lucose
fermentation. They were also characterised as positive in hydrogen
sulphide production, citrate uti l isation and decarboxylation of lysine
and ornithine. In contrast, they were negative for indole, urease,
phenylalanine and tryptophan.
Although this group of bacteria are ubiquitous in nature, some
are strictly host adapted. They have been known to be pathogenic to
humans causing enteric fever, gastroenteritis and septicaemia. These
pathogens also infect many animal species.
7
8
The systematics with respect to classification and nomenclature
of Salmonella have been in a state of flux for many years. The
classification authorities in different countries placed these bacteria in
different species or nomenclature that was inconsistent to the
international agreement. The existing taxonomic schemes based on
the biochemical characteristics and serology (Kaufmann, 1 966;
Edwards and Ewing 1 986; Le Minor, 1 992) have been traditional ly
attributed in the classification of this genus. In general practiceL these
bacteria were named either for the disease caused or animal involved.
For example, the human medical community embraced and used such
specific epithets as S. typhi, S. paratyphi etc. The veterinary medical
community were preferable to name the bacteria with the affected
animal species such as Salmonella choleraesuis, S. pollorum, S.
gallinarum, S. bovis morbificans etc. In addition, there were also
names associated to geographical areas such as S. arkansas, S.
birmingham, S. jurusalem, S. panama etc.
The Genus Salmonella
The Salmonella are natural inhabitants of the intestinal tract of
food animals including cattle, swine, sheep, chicken, turkeys and
ducks. This bacteria are also found in pet animals such as dogs, cats
and turtles. The wi ld animals l ike pigeon , repti les, m ice, rats and
insects are also natural reservoirs of the Salmonella. Usual ly,
9
disposals (faeces and urine) of the infected animals have been the
main sources of Salmonella spread to the environment and then
transmit the infection to other susceptible animals or human being.
This genus are classified into three main groups by World Health
Organisation (WHO) according to the host and mode of transmission
(Guthrie, 1 991 ) . The first group consisting of S. typhi, S. paratyphi A, B
and C , only associated to human infections. These serovars are
spread primarily either by food or water which have been d irectly or
indirectly contaminated by human waste. The second group is
considered to include serovars that are host-adapted in animals.
Included in this group are S. gallina rum adapted to poultry, S. dublin
adapted to catt le, S. abortus equi adapted to horse, S. abortus ovis
adapted to sheep, S. choleraesuis and S. typhisuis adapted to swine.
Some of this serovars particularly the dubl in and choleraesuis , are
pathogenic to human under certain condition. The third group are
serovars which show no particular host adaptation and are pathogenic
in either humans or animals. Including in this group are S. enteritidis
and S. typhimurium which cause the majority of salmonel los is
infection. Salmonel losis in humans caused by organisms from the
second and third group would be considered as zoonotic
salmonel losis.
1 0
Salmonella in Humans
Typhoid fever or enteric fever has plagued the human
populations for more than a century. Salmonella typhi, the etiologic
agent of typhoid fever was one of the earliest pathogenic bacteria to
be isolated and characterised (Le Minor, 1 992). This fever is an acute
i l lness causing bacteremic or septicemic infections. Paratyphoid fever
caused by S. paratyphi A, 8 or C, is pathological ly and clinical ly
s imi lar to typhoid, but i t forms mi lder i l lness. Typhoid infection in
humans is initiated by ingestion and survival of the bacteria in the
acidic pH of the stomach. With adequate numbers of infectious dose,
the bacteria penetrate the mucosa of the smal l intestine to the midlayer
where they are engulfed into the epithel ial cel ls . At this stage, the
bacteria wi l l cause inflammatory response in the small bowel and the
colon which then produced diarrhoea in the infected persons. A
systemic infection characterised by prolonged fever, headache,
enlargement of the spleen, rose spots on the abdominal surface,
constipation (50% of the cases) , diarrhoea (20% of cases) and muscle
soreness. The incubation period in such an infection may be as long
as 3 weeks fol lowing ingestion of the infectious dose of organisms. In
the past, the mortal ity rate in the untreated typhoid fever was as high
as 1 5%, but with antibiotic treatment this has been reduced to less
than 1 % (Guthrie, 1 991 ).